Real-time visualization of mutations and their fitness effects in single bacteria
| Autor: | Jean Ollion, Marina Elez, Lydia Robert |
|---|---|
| Přispěvatelé: | Laboratoire Jean PERRIN, Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Structure et évolution des génomes (SEG), CNS-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
DNA Replication
Computer science Computational biology dna medicine.disease_cause General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Mutation Accumulation 0302 clinical medicine Data acquisition medicine Escherichia coli [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology 030304 developmental biology 0303 health sciences Mutation Microbial Viability Escherichia coli Proteins DNA replication Equipment Design Microfluidic Analytical Techniques mutl Visualization Real time visualization mismatch repair Microscopy Fluorescence escherichia-coli DNA mismatch repair Fitness effects Single-Cell Analysis accumulation 030217 neurology & neurosurgery |
| Zdroj: | Nature Protocols Nature Protocols, Nature Publishing Group, 2019, 14 (11), pp.3126-3143. ⟨10.1038/s41596-019-0215-x⟩ Nature Protocols, 2019, 14 (11), pp.3126-3143. ⟨10.1038/s41596-019-0215-x⟩ |
| ISSN: | 1750-2799 |
| Popis: | Mutations are the driving force of evolution and the source of important pathologies. The characterization of the dynamics and effects of mutations on fitness is therefore central to our understanding of evolution and to human health. This protocol describes how to implement two methods that we recently developed: mutation visualization (MV) and microfluidic mutation accumulation (mu MA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (mu MA) to be followed directly in individual cells of Escherichia coli. MV provides a quantitative characterization of the dynamics of mutation occurrences, and mu MA allows precise estimation of the distribution of fitness effects (DFEs) of mutations. Both methods use microfluidics and time-lapse microscopy, and a fluorescent mismatch repair (MMR) MutL protein is used as a marker for nascent mutations. Here, we present a single protocol describing how to implement the MV and mu MA methods, including detailed procedures for microfluidic setup installation, data acquisition and data analysis and interpretation. Using this procedure, the microfluidic setup installation can be completed within 1 d, and automated data acquisition takes 2-4 d. |
| Databáze: | OpenAIRE |
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